This invention relates generally to novel azo bioconjugates for use in phototherapy.
The use of visible and near-infrared (NIR) light in clinical practice is growing rapidly. Compounds absorbing or emitting in the visible, NIR, or long-wavelength (UV-A,  greater than 350 nm) region of the electromagnetic spectrum are potentially useful for optical tomographic imaging, endoscopic visualization, and phototherapy. However, a major advantage of biomedical optics lies in its therapeutic potential. Phototherapy has been demonstrated to be a safe and effective procedure for the treatment of various surface lesions, both external and internal. Its efficacy is comparable to that of radiotherapy, but without the harmful radiotoxicity of critical non-target organs.
Phototherapy has been in existence for many centuries and has been used to treat various skin surface ailments. As early as 1400 B.C. in India, plant extracts (psoralens), in combination with sunlight, were used to treat vitiligo. In 1903, Von Tappeiner and Jesionek used eosin as a photosensitizer for the treatment of skin cancer, lupus of the skin, and condylomata of female genitalia. Over the years, the combination of psoralens and ultraviolet A (low-energy) radiation has been used to treat a wide variety of dermatological diseases including psoriasis, parapsoriasis, cutaneous T-cell lymphoma, eczema, vitiligo, areata, and neonatal bilirubinemia. Although the potential of cancer phototherapy has been recognized since early 1900""s, systematic studies to demonstrate safety and efficacy began only in 1967 with the treatment of breast carcinoma. Dougherty et al. subsequently conclusively established that long-term cure is possible with photodynamic therapy (PDT). Currently, phototherapeutic methods are also being investigated for the treatment of some cardiovascular disorders such as atherosclerosis and vascular restenosis for the treatment rheumatoid arthritis, and for the treatment of some inflammatory diseases such as Crohn""s disease.
Phototherapeutic procedures require photosensitizers (i.e. chromophores) which have high absorptivity. These compounds should preferably be chemically inert, and become activated only upon irradiation with light of an appropriate wavelength. Light-initiated selective tissue injury can be induced when these photosensitizers bind to target tissues, either directly or through attachment to a bioactive carrier. Furthermore, if the photosensitizer is also a chemotherapeutic agent (e.g. anthracycline antitumor agents), then an enhanced therapeutic effect can be attained.
Effective phototherapeutic agents should have the following properties: (a) large molar extinction coefficient; (b) long triplet lifetime; (c) high yield of singlet oxygen and/or other reactive intermediates, viz., free radicals, nitrenes, carbenes, open-shell ionic species such as cabonium ions and the like; (d) efficient energy or electron transfer to cellular components; (e) low tendency to form aggregation in aqueous milieu; (f) efficient and selective targeting of lesions; (g) rapid clearance from blood and non-target tissues; (h) low systemic toxicity; and (i) lack of mutagenicity.
Photosensitizers operate via two distinct pathways, termed Types 1 and 2. The type 1 mechanism is shown in the following scheme: 
After photoexcitation, the Type 1 mechanism involves direct energy or electron transfer from the photosensitizer to the cellular components, thereby causing cell death. After photoexcitation, the Type 2 mechanism involves distinct steps as shown in the following scheme: 
In the first step, singlet oxygen is generated by energy transfer from the triplet excited state of the photosensitizer to the oxygen molecules surrounding the tissues. In the second step, collision of a singlet oxygen with the tissues promotes tissue damage. In both Type 1 and Type 2 mechanisms, the photoreaction proceeds via the lowest triplet state of the sensitizer. Hence, a relatively long triplet lifetime is required for effective phototherapy. In contrast, a relatively short triplet lifetime is required to avoid photodamage to the tissue caused by photosensitizers.
The biological basis of tissue injury brought about by tumor phototherapeutic agents has been the subject of intensive study. Various reasonable biochemical mechanisms for tissue damage have been postulated even though the type and number of photosensitizers employed in these studies are relatively small. These biochemical mechanisms are as follows: a) cancer cells upregulate the expression of low density lipoprotein (LDL) receptors, and PDT agents bind to LDL and albumin selectively; (b) porphyrin-like substances are selectively taken up by proliferative neovasculature; (c) tumors often contain an increased number of lipid bodies and are thus able to bind to hydrophobic photosensitizers; (d) a combination of xe2x80x9cleakyxe2x80x9d tumor vasculature and reduced lymphatic drainage causes porphyrin accumulation; (e) tumor cells may have increased capabilities for phagocytosis or pinocytosis of porphyrin aggregates; (f) tumor associated macrophages may be largely responsible for the concentration of photosensitizers in tumors; and (g) cancer cells may undergo apoptosis induced by photosensitizers. Among these mechanisms, (f) and (g) are the most general and, of these two alternatives, there is a general consensus that (f) is the most likely mechanism by which the phototherapeutic effect of porphyrin-like compounds is induced.
Most of the currently known photosensitizers are commonly referred to as PDT agents and operate via the Type 2 mechanism. For example, Photofrin II, a hematoporphyrin derivative, was approved by the United States Food and Drug Administration for the treatment of bladder, esophageal, and late-stage lung cancers. However, Photofrin II has been shown to have several drawbacks: low molar absorptivity, (xcex5=3000Mxe2x88x921), low singlet oxygen quantum yield (xcfx86=0.1), chemical heterogeneity, aggregation, and prolonged cutaneous photosensitivity. Hence, there has been considerable effort in developing safer and more effective photosensitizers for PDT that exhibit improved light absorbance properties, better clearance, and decreased skin photosensitivity compared to those of Photofrin II. These photosensitizers include monomeric porphyrin derivatives, corrins, cyanines, phthalocyanines, phenothiazines, rhodamines, hypocrellins, and the like. However, these phototherapeutic agents also mainly operate via the Type 2 mechanism.
Surprisingly, there has not been much attention directed at developing Type 1 phototherapeutic agents, despite the fact that the Type 1 mechanism seems inherently more efficient than the Type 2 mechanism. First, unlike Type 2, Type 1 photosensitizers do not require oxygen for causing cellular injury. Second, the Type 1 mechanism involves two steps (photoexcitation and direct energy transfer) whereas the Type 2 mechanism involves three steps (photoexcitation, singlet oxygen generation, and energy transfer). Furthermore, some tumors have hypoxic regions that render the Type 2 mechanism ineffective. In spite of the drawbacks associated with the Type 2 mechanism, however, only a small number of compounds have been developed that operate through the Type 1 mechanism, e.g. anthracyline antitumor agents.
Thus, there is a need to develop effective phototherapeutic agents that operate through the Type 1 mechanism. Phototherapeutic efficacy can be further enhanced if the excited state photosensitizers can generate reactive intermediates such as free radicals, nitrenes, carbenes, and the like. These have much longer lifetimes than the excited chromophore and have been shown to cause considerable cell injury.
The present invention addresses this need and discloses novel azo derivatives and their bioconjugates that absorb in the low-energy, ultraviolet, visible, or near-infrared (NIR) region of the electromagnetic spectrum that are used for the phototherapy of tumors and other lesions. More specifically, the present invention discloses azo compounds having the formula 1
where Q is a single bond or xe2x80x94CR1R2; R1 and R2 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C10 aryl, C1-C10 alkoxyalkyl; C1-C10 polyhydroxyalkyl, xe2x80x94(CH2)aCO2H, and xe2x80x94(CH2)bNR3R4; R3 and R4 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C10 aryl, C1-C10 polyhydroxyalkyl, and xe2x80x94(CH2)aCO2H; R5, R6, and R7 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C10 aryl, hydroxyl, xe2x80x94SO3H, C1-C10 alkoxyl, C1-C10 polyhydroxyalkyl, C1-C10 polyalkoxyalkyl, xe2x80x94(CH2)aCO2H, and xe2x80x94(CH2)bNR3R4; X is selected from the group consisting of xe2x80x94CR8R9, xe2x80x94Oxe2x80x94, xe2x80x94NR3, xe2x80x94Sxe2x80x94, and xe2x80x94Cxe2x95x90O; Y is selected from the group consisting of xe2x80x94CR10R11, xe2x80x94Oxe2x80x94, xe2x80x94NR3, xe2x80x94Sxe2x80x94, and xe2x80x94Cxe2x95x90O; Z is selected from the group consisting of xe2x80x94CR12R13, xe2x80x94Oxe2x80x94, xe2x80x94NR3, xe2x80x94Sxe2x80x94, and xe2x80x94Cxe2x95x90O; R8 to R13 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C10 aryl, C1-C10 alkoxyalkyl, C1-C10 polyhydroxyalkyl, xe2x80x94(CH2)aCO2H, and xe2x80x94(CH2)bNR3R4; R5-R6, R6-R7, R8-R10, or R10-R12 together optionally form a six-membered alicyclic or aromatic ring; E is either a hydrogen atom or is selected from the group consisting of antibodies, peptides, peptidomimetics, carbohydrates, glycomimetics, drugs, hormones, or nucleic acids; L is a linker unit selected from the group consisting of xe2x80x94(CH2)cxe2x80x94, xe2x80x94(CH2)dCONR3xe2x80x94, xe2x80x94N(R3)CO(CH2)dxe2x80x94, xe2x80x94OCO(CH2)exe2x80x94, xe2x80x94(CH2)fCO2xe2x80x94, xe2x80x94OCONHxe2x80x94, xe2x80x94OCO2xe2x80x94, xe2x80x94HNCONHxe2x80x94, xe2x80x94HNCSNHxe2x80x94, xe2x80x94HNNHCOxe2x80x94, xe2x80x94OSO2xe2x80x94, xe2x80x94NR3(CH2)gCONR4xe2x80x94, xe2x80x94CONR3(CH2)hNR4COxe2x80x94, and xe2x80x94NR3CO(CH2)iCONR4; a, b, d to i independently range from 0 to 10, and c ranges from 1 to 10.
The present invention also discloses a method of performing a phototherapeutic or photodiagnostic procedure using the inventive azo compounds and their derivatives. In the method, an effective amount of an azo photosensitizer having the formula 1
is administered to a subject; where Q is a single bond or xe2x80x94CR1R2; R1 and R2 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C10 aryl, C1-C10 alkoxyalkyl, C1-C10 polyhydroxyalkyl, xe2x80x94(CH2)aCO2H, and xe2x80x94(CH2)bNR3R4; R3 and R4 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C10 aryl, C1-C10 polyhydroxyalkyl, and xe2x80x94(CH2)aCO2H; R5, R6, and R7 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C10 aryl, hydroxyl, xe2x80x94SO3H, C1-C10 alkoxyl, C1-C10 polyhydroxyalkyl, C1-C10 polyalkoxyalkyl, xe2x80x94(CH2)aCO2H, and xe2x80x94(CH2)bNR3R4; X is selected from the group consisting of xe2x80x94CR8R9, xe2x80x94Oxe2x80x94, xe2x80x94NR3, xe2x80x94Sxe2x80x94, and xe2x80x94Cxe2x95x90O; Y is selected from the group consisting of xe2x80x94CR10R11, xe2x80x94Oxe2x80x94, xe2x80x94NR3, xe2x80x94Sxe2x80x94, and xe2x80x94Cxe2x95x90O; Z is selected from the group consisting of xe2x80x94CR12R13, xe2x80x94Oxe2x80x94, xe2x80x94NR3, xe2x80x94Sxe2x80x94, and xe2x80x94Cxe2x95x90O; R8 to R13 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C10 aryl, C1-C10 alkoxyalkyl, C1-C10 polyhydroxyalkyl, xe2x80x94(CH2)aCO2H, and xe2x80x94(CH2)bNR3R4; R5-R6, R6-R7, R8-R10, or R10-R12 together optionally form a six-membered ring; E is either a hydrogen atom or is selected from the group consisting of antibodies, peptides, peptidomimetics, carbohydrates, glycomimetics, drugs, hormones, or nucleic acids; L is a linker unit selected from the group consisting of xe2x80x94(CH2)cxe2x80x94, xe2x80x94(CH2)dCONR3xe2x80x94, xe2x80x94N(R3)CO(CH2)dxe2x80x94, xe2x80x94OCO(CH2)exe2x80x94, (CH2)fCO2xe2x80x94, xe2x80x94OCONHxe2x80x94, xe2x80x94OCO2xe2x80x94, xe2x80x94HNCONHxe2x80x94, xe2x80x94HNCSNHxe2x80x94, xe2x80x94HNNHCOxe2x80x94, xe2x80x94OSO2xe2x80x94, xe2x80x94NR3(CH2)gCONR4xe2x80x94, xe2x80x94CONR3(CH2)hNR4COxe2x80x94, and xe2x80x94NR3CO(CH2)iCONR4; a, b, d to i independently range from 0 to 10, and c ranges from 1 to 10. The compound is photoactivated and a phototherapeutic or photodiagnostic procedure for tumors, impaired vasculature or other lesions is subsequently performed.
For targeting purposes, external attachment of an epitope is used unless the azo compounds themselves preferentially accumulate in the target tissue. For example, if the photosensitizing chromophore is an anthracycline moiety, it can bind to cancer cells directly and may not require an epitope for targeting purposes.
These and other advantages and embodiments of the inventive compounds and methods will be apparent in view of the following figures, description, and example.